1. Field of the Invention
This invention relates to polyamides which bind to predetermined sequences in the minor groove of double stranded DNA.
2. Description of the Related Art
The design of synthetic ligands that read the information stored in the DNA double helix has been a long standing goal of chemistry. Cell-permeable small molecules which target predetermined DNA sequences are useful for the regulation of gene-expression. Oligodeoxynucleotides that recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity. Although oligonucleotides and their analogs have been shown to interfere with gene expression, the triple helix approach is limited to purine tracks and suffers from poor cellular uptake. The. development of pairing rules for minor groove binding polyamides derived from N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids provides another code to control sequence specificity. An Im/Py pair distinguishes Gxc2x7C from Cxc2x7G and both of these from Axc2x7T or Txc2x7A base pairs. Wade, W. S., Mrksich, M. and Dervan, P. B. describes the design of peptides that bind in the minor groove of DNA at 5xe2x80x2-(A,T)G(A,T)C(A,T)-3xe2x80x2 sequences by a dimeric side-by-side motif. J. Am. Chem. Soc. 114, 8783-8794 (1992); Mrksich, M. et al. describes antiparallel side-by-side motif for sequence specific-recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamidenetropsin. Proc. Natl. Acad. Sci. USA 89, 7586-7590 (1992); Trauger, J. W., Baird, E. E. Dervan, P. B. describes the recognition of DNA by designed ligands at subnanomolar concentrations. Nature 382, 559-561 (1996). A Py/Py pair specifies Axc2x7T from Gxc2x7C but does not distinguish Axc2x7T from Txc2x7A. Pelton, J. G. and Wemmer, D. E. describes the structural characterization of a 2-1 distamycin A-d(CGCAAATTTGGC) complex by two-dimensional NMR. Proc. Natl. Acad. Sci. USA 86, 5723-5727 (1989); White, S., Baird, E. E. and Dervan, P. B. Describes the effects of the Axc2x7T/Txc2x7A degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of DNA. Biochemistry 35, 6147-6152 (1996); White, S., Baird, E. E. and Dervan, P. B. describes the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides. Chem. and Biol. 4, 569-578 (1997); White, S., Baird, E. E. and Dervan, P. B. describes the 5xe2x80x2-3xe2x80x2 N-C orientation preference for polyamide binding in the minor groove. In order to break this degeneracy, a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp) incorporated into a polyamide and paired opposite Py, has been found to discriminate Axc2x7T from Txc2x7A. The replacement of a single hydrogen atom on the pyrrole with a hydroxy group in a Hp/Py pair regulates affinity and specificity of a polyamide by an order of magnitude. Utilizing Hp together with Py and Im in polyamides to form four aromatic amino acid pairs (Im/Py, Py/Im, Hp/Py, and Py/Hp) provides a code to distinguish all four Watson-Crick base pairs in the minor groove of DNA.
The invention encompasses improved polyamides for binding to the minor groove of double stranded (xe2x80x9cduplexxe2x80x9d) DNA. The polyamides are in the form of a hairpin comprising two groups of at least three consecutive carboxamide residues, the two groups covalently linked by an aliphatic amino acid residue, preferably y-aminobutyric acid or 2,4 diaminobutyric acid, the consecutive carboxamide residues of the first group pairing in an antiparallel manner with the consecutive carboxamide residues of the second group in the minor groove of double stranded DNA. The improvement relates to the inclusion of a binding pair of Hp/Py carboxamides in the polyamide to bind to a Txc2x7A base pair in the minor groove of double stranded DNA or Py/Hp carboxamide binding pair in the polyamide to bind to an Axc2x7T base pair in the minor groove of double stranded DNA. The improved polyamides have at least three consecutive carboxamide pairs for binding to at least three DNA base pairs in the minor groove of a duplex DNA sequence that has at least one Axc2x7T or Txc2x7A DNA base pair, the improvement comprising selecting a Hp/Py carboxamide pair to correspond to a Txc2x7A base pair in the minor groove or a Py/Hp carboxamide pair to bind to an Axc2x7T DNA base pair in the minor groove. Preferably the binding of the carboxamide pairs to the DNA base pairs modulates the expression of a gene.
In one preferred embodiment, the polyamide includes at least four consecutive carboxamide pairs for binding to at least four base pairs in a duplex DNA sequence. In another preferred embodiment, the polyamide includes at least five consecutive carboxamide pairs for binding to at least five base pairs in a duplex DNA sequence. In yet another preferred embodiment, the polyamide includes at least six consecutive carboxamide pairs for binding to at least six base pairs in a duplex DNA sequence. In one preferred embodiment, the improved polyamides have four carboxamide binding pairs that will distinguish Axc2x7T, TEA, Cxc2x7G and Gxc2x7C base pairs in the minor groove of a duplex DNA sequence. The duplex DNA sequence can be a regulatory sequence, such as a promoter sequence or an enhancer sequence, or a gene sequence, such as a coding sequence or a non-coding sequence. Preferably, the duplex DNA sequence is a promoter sequence.
The preparation and the use of polyamides for binding in the minor groove of double stranded DNA are extensively described in the art. This invention is an improvement of the existing technology that uses 3-hydroxy-N-methylpyrrole to provide carboxamide binding pairs for DNA binding polyamides.
The invention encompasses polyamides having xcex3-aminobutyric acid or a substituted xcex3-aminobutyric acid to form a hairpin with a member of each carboxamide pairing on each side of it. Preferably the substituted xcex3-aminobutyric acid is a chiral substituted xcex3-aminobutyric acid such as (R)-2,4-diaminobutyric acid. In addition, the polyamides may contain an aliphatic amino acid residue, preferably a xcex2-alanine residue, in place of a non-Hp carboxamide. The xcex2-alanine residue is represented in formulas as xcex2. The xcex2-alanine residue becomes a member of a carboxamide binding pair. The invention further includes the substitution as a xcex2xc2x7xcex2 binding pair for non-Hp containing binding pair. Thus, binding pairs in addition to the Hp/Py and Py/Hp are Im/xcex2, xcex2/Im, Py/xcex2, xcex2/Py, and xcex2/xcex2.
The polyamides of the invention can have additional moieties attached covalently to the polyamide. Preferably the additional moieties are attached as substituents at the amino terminus of the polyamide, the carboxy terminus of the polyamide, or at a chiral (R)-2,4-diaminobutyric acid residue. Suitable additional moieties include a detectable labeling group such as a dye, biotin or a hapten. Other suitable additional moieties are DNA reactive moieties that provide for sequence specific cleavage of the duplex DNA.